The invention relates generally to platforms for the development of assays for the simultaneous detection of multiple bacteria and, more specifically, the use of the T4 bacteriophage, and related T even bacteriophages, in assays for the simultaneous detection of human pathogenic bacteria.
Bacteriophage within the T even family infect many diverse bacteria, including Escherichia coli, Salmonella spp., Shigella spp., Yersinia spp., Aeromonas spp., Burkholderia spp., Pseudomonas spp., Acinebacter spp., Vibrio spp., Klebsiella, spp., Citrobacter spp., Proteus spp., and Serratia spp. These bacteria are all important as causes of food spoilage, and human and animal illness. Many of these pathogens (E. coli, Salmonella, Shigella, Aeromonas, Vibrio) cause serious foodborne illness in humans. Foodborne illness accounts for 75 million illnesses in the U.S. each year. In addition, several of the pathogens described above (Yersinia pestis, Burkholderia spp.) are considered as Category A select biological agents, capable to be used as biological weapons. Therefore rapid and simple detection methods are needed to ensure the safety of the public from these pathogens.
In traditional reporter bacteriophage assays, a temperate bacteriophage is screened against many different bacteria to determine its host range, followed by genome sequencing, and creation of a genetically modified bacteriophage. This process typically takes years, and needs to be repeated for every new assay and bacteriophage. Additionally, the methods used to create the genetically modified bacteriophage typically resulted in the incorporation of an antibiotic resistance gene into the bacteriophage chromosome, which is not advantageous since it is possible for the genetically engineered temperate bacteriophage to transfer the antibiotic resistance gene to its host.
The method described in this invention eliminates many of the traditional reporter bacteriophage creation steps, uses a lytic bacteriophage (lytic bacteriophage kill their host, so there is no worry about transferring virulence genes to the host), does not incorporate an antibiotic resistance gene into the bacteriophage chromosome, and allows for the creation of multiple detection assays within a matter of weeks to months as opposed to years. This also significantly decreases the cost of development.
The advantages of this system over other systems are numerous. Since bacteriophage are only capable of growing in viable bacteria, the reporter bacteriophages used in this system are capable of distinguishing between viable and non-viable bacteria. This is a major advantage over conventional PCR assays and ELISA techniques. In addition, the technique requires only the addition of the reporter bacteriophage to the sample of cells (the cells can be isolated from complex sample matrices using standard immunomagnetic separation techniques or other methods), followed by assaying for the reporter protein. This significantly decreases the labor intensiveness of the method, as compared to other methods like PCR and ELISA. The use of a β-galactosidase gene in this method allows for calorimetric detection of the signal, making the test instrument-free, which is another advantage over conventional rapid microbiological detection methods. Finally, the method described in this invention, in which multiple assays can be produced from a single bacteriophage, is advantageous and cost effective because the assays can be produced based on a standardized platform, without the need to completely genetically and phenotypically characterize new bacteriophage for every new test.
One disadvantage of the currently available reporter bacteriophage assays is the need for an instrument to detect the reporter protein of interest, which limits the ability of these methods to be used in the field. The present invention also includes a single-tube apparatus hat employs the reporter bacteriophage platform in an assay that is simple and easy to perform, inexpensive and fast. This reporter bacteriophage assay is self-enclosed, and detected using a choice of substrates. One class of these substrates (calorimetric substrates) allows for the production of a visible colorimetric product, eliminating the need for instrument based detection.